Modulation of gene expression by endogenous, non-coding RNAs have been increasingly appreciated and known to play a role in eukaryotic development, and epigenetic control. Recently, methods have been developed to trigger RNA interference (RNAi) against specific targets in mammalian cells by introducing exogenously produced or intra-cellularly expressed small interfering RNA (siRNA) molecules.
These quick, inexpensive and effective methods have proven to be effective for knockdown experiments in vitro and in vivo. The ability to attain such selective gene silencing has led to the hypothesis that siRNAs can be used to suppress gene expression for therapeutic benefit. The ideal candidates for such an siRNA approach would be dominantly inherited diseases.
Recent studies by Miller V et al1 have shown that siRNAs can be used to target untreatable neurodegenerative diseases such as polyglutamine (polyQ) neurodegeneration in Machado-Joseph disease, spinocerebellar ataxia type 3 (MJDSCA3) and Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). These studies have focused exclusively on selective silencing of the transcript produced by the mutant allele1. RNA interference has proven to be an efficient strategy for silencing mutant tau allele V337M, however, selective depletion of mutant allele was not completely achieved in the study because there was a partial depletion of the wild type allele of tau1. More recently, another example of MAPT-targeting siRNAs was described2, wherein a mix of four siRNAs aimed at suppressing all tau isoforms in a mouse model of tauopathy was tested.
The MAPT (Microtubule associated protein tau) gene consists of 16 exons and its expression is regulated by complex alternative splicing. This results in the production of two types of alternatively spliced transcripts: one bearing Exon 10, also known as 4R (Four microtubule repeats) isoform and the other that lacks Exon 10 is called 3R isoform (Three microtubule repeats). Equal levels of these two isoforms are expressed in normal human adult brain. Though several mutations causing FTDP-17 are known in MAPT, a half of these affect alternative splicing of Exon 10. These include mis-sense mutations, silent mutations and point mutations which are located in Exon 10, introns 9 and 10. They are known to implicate an increase in Exon 10 causing an excessive accumulation of 4R. This leads to the formation of neurofibrillary tangles, hence resulting in neurodegeneration.
It is worth mentioning that abnormalities of tau are linked to the pathogenesis of neurodegenerative disease collectively termed as “tauopathies”, and significantly elevated levels of tau are present in AD (Alzheimer's disease) brains.
A few approaches have been used for the correction of Exon 10 inclusion in FTDP-17:                Small molecules: a screening has been performed which yielded cardiotonic steroids as exon 10 splicing modulator, albeit non-specific, drugs3, and        Antisense oligonucleotides for exon skipping: US-A-2003/0170704 by Stamm et al. relates to substances which are capable of controlling the inclusion of MAPT exon 10 (proteic splicing regulators or their cDNA, polypeptides controlling the phosphorylation of splicing regulators, or their cDNA, and antisense oligonucleotides which interact with the splice junctions of MAPT exon 10). Moreover, work by Kalbfuss et al.4 has demonstrated that oligoribonucleotides binding to Exon 10 splicing junctions could suppress the predominant inclusion of Exon 10 in tau mRNA in the context of rat PC12 cells. However, recent work by Sud R et al5 showed that targeting Exon 10 with antisense morpholino oligonucleotides did not yield exon-skipping in neuroblastoma cell lines. These authors claim that splicing regulation of exon 10 in cells expressing predominantly 4R isoform may vary from the neuroblastoma cell line in which the 3R isoform dominates.        Trans-splicing of exon 10: RNA reprogramming using spliceosome-mediated RNA trans-splicing (SMaRT) was used to correct aberrant Exon 10 splicing resulting from FTDP-17 mutations in a minigene system in cells in culture6. This approach, however, is affected by low efficiency.        